With respect to patients with myocardial infarction or cerebral infarction, warfarin is used widely as a drug for preventing blood coagulation. The suitable dose of warfarin differs considerably depending on the race and further there are differences among individuals even though they are of the same race. When a large amount of warfarin is administered, there is a risk of causing, for example, epistaxis or internal hemorrhage in the skin as well as side effects such as intracerebral hemorrhage in some cases. Accordingly, it is very important to determine the suitable dose of warfarin for each patient individually in the treatment.
In determining such a suitable dose value of warfarin, recently, it has been reported that polymorphisms of the CYP2C9 gene and the VKORC1 gene are associated with the drug action of warfarin (Nonpatent Document 1 and Nonpatent Document 2). The CYP2C9 gene is a gene that codes for cytochrome P450, which produces a warfarin metabolic enzyme. The VKORC1 gene is a gene that codes for protein, which acts on vitamin K involved in blood coagulation. Therefore to detect polymorphisms of these two genes also is very important for determining the suitable dose of warfarin for each patient to reduce the side effects.
On the other hand, detection of a point mutation, a so-called single nucleotide polymorphism (SNP), is performed widely as a method of analyzing, at the gene level, for example, the causes of all types of diseases and the individual differences in disease liability (susceptibility to diseases) and in drug action. Examples of common methods of detecting a point mutation include: (1) a direct sequencing method in which the region corresponding to a sequence to be subjected to detection in a target DNA of a sample is amplified by polymerase chain reaction (PCR) and all the gene sequences thereof are analyzed, (2) RFLP analysis in which the region corresponding to a sequence to be subjected to detection in a target DNA of a sample is amplified by PCR, the amplification product thus obtained is cut with a restriction enzyme whose cleaving action differs depending on the presence or absence of the target mutation in the sequence to be subjected to detection and is then electrophoresed, and thereby typing is performed, and (3) an ASP-PCR method in which PCR is performed using a primer with a target mutation located in the 3′-end region and the mutation is judged depending on the presence or absence of amplification.
However, since these methods require, for example, purification of DNA extracted from a sample, electrophoresis, and a treatment with a restriction enzyme, they take time and cost. Furthermore, after PCR is performed, it is necessary to open the reaction container once. Accordingly, there is a possibility that the amplification product may contaminate the next reaction system and thereby the analysis accuracy may be deteriorated. Moreover, since it is difficult to automate, multiple samples cannot be analyzed. Further, the aforementioned ASP-PCR method (3) has lower specificity, which also is a problem.
Because of these problems, recently, a method of analyzing the melting temperature (Tm) of double-stranded nucleic acid formed of a probe and target nucleic acid is used as a method of detecting a point mutation. Since such a method is performed through, for example, Tm analysis or analysis of the melting curve of a double strand, it is referred to as melting curve analysis. This method is described below. That is, first, a probe complementary to a sequence to be subjected to detection containing a point mutation to be detected is used to form a hybrid (double-stranded DNA) between the aforementioned probe and a target single-stranded DNA contained in a detection sample. Subsequently, this hybridization product is heat-treated, and dissociation (melting) of the hybrid accompanying the temperature rise is detected by a change in a signal of, for example, absorbance. The Tm value then is determined based on the result of the detection and the presence or absence of any point mutation is judged accordingly. The higher the homology of the hybridization product, the higher the Tm value, and the lower the homology, the lower the Tm value. Therefore the Tm value (reference value for assessment) is determined beforehand with respect to the hybridization product between the sequence to be subjected to detection containing a point mutation and a probe complementary thereto, and then the Tm value (measured value) of the hybridization product between the target single-stranded DNA contained in the detection sample and the aforementioned probe is measured. When the measured value is comparable to the reference value, it is considered as matching, that is, it can be judged that a point mutation is present in the target DNA. On the other hand, when the measured value is lower than the reference value, it is considered as mismatching, that is, it can be judged that no point mutation is present in the target DNA. Furthermore, according to this method, it also is possible to automate gene analysis.
However, such a detection method using Tm analysis also has a problem in that plural sequences cannot be analyzed in one reaction solution as in the cases of the aforementioned analysis methods (1) to (3). As described above, a polymorphism of the CYP2C9 gene and a polymorphism of the VKORC1 gene are associated with the effect of warfarin. Accordingly, it is desirable to analyze the polymorphisms of both the genes and to determine comprehensively the prescription of warfarin based on the results of the analyses. However, since isozymes are present in the respective genes, there is a possibility that genes coding for isozymes other than the target genes also are amplified in PCR. In conventional methods, therefore, for example, in order to check the respective polymorphisms of the CYP2C9 gene and the VKORC1 gene, it was necessary to amplify target regions of the respective genes in different reaction systems, respectively, and to analyze the resultant amplification products separately. As described above, in conventional methods, it is very difficult to allow only these two genes to serve as templates and to amplify specifically only the respective target regions of the respective genes. Furthermore, as described above, since even the analysis of one sample is accompanied by a considerable amount of time and energy, it is not practical to analyze multiple samples, which is a problem.    [Nonpatent Document 1] Simone Rost et al., Nature Vol. 427 2004 letters to nature    [Nonpatent Document 2] Mark J. Rieder et al., The New England Journal of Medicine 352; 22, 2005